Fig 2.

Immunoblots of 4-HNE modified proteins in liver mitochondria incubated with 4-HNE. Freshly isolated liver mitochondria (0.5 mg) were incubated with 0, 10, or 50 µM 4-HNE for 10 min to generate samples containing 4-HNE adducted proteins. After incubations, samples were flash frozen in liquid N₂ and stored at −80 °C until used in electrophoresis experiments. Panel A: Mitochondrial protein (20 µg) was separated on 10% SDS-PAGE gels and immunoblotted to detect 4-HNE modified proteins. Results show increased in 4-HNE modified proteins in mitochondria incubated with 50 µM 4-HNE. A small increase in 4-HNE labeling is observed at 10 µM 4-HNE. The five 4-HNE modified proteins detected in mitochondria from control and ethanol-fed rats (Fig. 1) are highlighted here by the arrows. A slightly different pattern in separation is observed here as samples were separated using 10% mini-gels compared to an 8–20% large-format gradient gels (Fig. 1). Panel B: Protein (100 µg) from mitochondria treated with 0 and 50 µM 4-HNE was separated by 2D IEF SDS-PAGE and immunoblotted to visualize 4-HNE modified proteins. Results show an increase in the number of 4-HNE modified proteins in mitochondria exogenously treated with 50 µM 4-HNE as compared to untreated mitochondria.